Generation of pRSAD2 gene knock-in pig via CRISPR/Cas9 technology

A wide range of endemic and epidemic viruses, including classic swine fever virus (CSFV), pseudorabies virus (PRV) and others, are among the most economically important pathogens in pigs and have severely affected the national economy, human health and animal welfare and productivity. The RSAD2 exhibits antiviral activity against various DNA and RNA viruses. In this study, we successfully accomplished site-specific insertion of the porcine RSAD2 gene (pRSAD2) at the porcine ROSA26 (pROSA26) locus, generating pRSAD2 gene knock-in (pRSAD2-KI) PK-15 cells and porcine foetal fibroblasts (PFFs) via CRISPR/Cas9 technology. Gene expression analysis confirmed that pRSAD2-KI cells stably and efficiently overexpressed the pRSAD2 gene. Furthermore, viral challenge studies in vitro indicated that site-specific integration of the pRSAD2 gene not only effectively reduced CSFV infection but also PRV infection. More importantly, we ultimately successfully produced a pRSAD2-KI pig that constitutively overexpressed the pRSAD2, viral challenge results indicated that fibroblasts isolated from the pRSAD2-KI pig reduced CSFV infection. Taken together, these results suggest that CRISPR/Cas9-mediated knock-in strategy can be used for producing pRSAD2-KI pigs.